PROJECT SUMMARY We have met or exceeded the Phase I success criteria; developed the capture free ligation-based TempO- Seq gene expression assay; implemented, validated, and offered early access to the NIEHS TempO-Seq rat surrogate assay for profiling RNA, cell lysates, and lysates of formalin fixed, paraffin embedded (FFPE) tissue as small as 0.01mm3; and shown that the NIEHS human S1500+ TempO-Seq assay measures differential expression that is highly correlated to RNAseq and identified prostate cancer biomarkers and therapeutic targets from archived prostate FFPE. This Phase II program will address an NIEHS area of special interest Topic (A.) ?Development and validation of alternative test methods to protect human and animal health while reducing, refining, or replacing animal tests?. We will validate the use of the rat whole transcriptome assay and a human whole transcriptome assay in which the NIEHS S1500 rate surrogate is embedded for profiling archived rat and clinical FFPE samples. We will confirm that the surrogate assay data can be used in silico to accurately predict gene expression changes across the whole transcriptome, though only a few thousand genes are directly measured. We will demonstrate that the TempO-Seq whole transcriptome assays can profile many more samples than RNAseq for the same cost, and that the surrogate assay lowers cost even further. Consequently, the TempO-Seq assay of FFPE will permit the archives of animal FFPE to be mined cost effectively, providing in vivo molecular phenotypes for compounds tested in the past, without having to repeat animal studies. We will demonstrate this by profiling archived FFPE provided by NIEHS from several toxicity and dietary studies for which there is associated RNAseq, microarray, and/or histology to correlate with TempO-Seq results. We showed in Phase I that the precision of the TempO-Seq assay provides ?between animal? average CVs of <10%, and that this precision enables identification of molecular signatures for differential ED50s. This will improve the ability of medicinal chemists to increase the therapeutic safety window of drugs, and will be useful for validating in vitro assays for use in order to spare animal studies. It will also enable toxicity to be detected at lower doses and earlier time points, thus permitting the size of animal studies to be reduced or discontinuing compounds early before investment in long term animal models, significantly reducing clinical safety testing. By dosing rats with reference compounds and correlating histology to TempO-Seq data, we will demonstrate that precise ED50s for genes and pathway signatures can be established. Clinical utility will be demonstrated from a translational study of archived FFPE from breast cancer patients for which 5-, 10-, 20-year outcomes are known, and from which molecular phenotypes of breast cancer subtypes and prognostic biomarkers will be identified, confirming and extending what is known today.